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The molecular mechanisms controlling the balance of quiescence and proliferation in adult neural stem cells (NSCs) are often deregulated in brain cancers such as glioblastoma (GBM). Previously, we reported that FOXG1, a forebrain-restricted neurodevelopmental transcription factor, is frequently upregulated in glioblastoma stem cells (GSCs) and limits the effects of cytostatic pathways, in part by repression of the tumour suppressor Foxo3. Here, we show that increased FOXG1 upregulates FoxO6, a more recently discovered FoxO family member with potential oncogenic functions. Although genetic ablation of FoxO6 in proliferating NSCs has no effect on the cell cycle or entry into quiescence, we find that FoxO6-null NSCs can no longer efficiently exit quiescence following FOXG1 elevation. Increased FoxO6 results in the formation of large acidic vacuoles, reminiscent of Pak1-regulated macropinocytosis. Consistently, Pak1 expression is upregulated by FOXG1 overexpression and downregulated upon FoxO6 loss in proliferative NSCs. These data suggest a pro-oncogenic role for FoxO6, downstream of GBM-associated elevated FOXG1, in controlling quiescence exit, and shed light on the potential functions of this underexplored FoxO family member., (© 2024. Published by The Company of Biologists Ltd.)

Background and Aims: Epidemiological studies have shown that subnormal levels of vitamin D (25[OH]D) are associated with a more aggravated clinical course of ulcerative colitis [UC]. Despite an increased focus on the therapeutic importance of vitamin D and vitamin D receptor [VDR] signalling, the mechanisms underlying the effects of the vitamin D-VDR axis on UC remain elusive. Therefore, we aimed to investigate whether exposure to active vitamin D (1,25[OH]2D3/VDR) signalling in human organoids could influence the maintenance of the colonic epithelium., Methods: Intestinal VDR expression was studied by immunohistochemistry, RNA expression arrays, and single-cell RNA sequencing of colonic biopsy specimens obtained from patients with UC and healthy individuals. To characterise the functional and transcriptional effects of 1,25[OH]2D3, we used patient-derived colonic organoids. The dependency of VDR was assessed by knocking out the receptor with CRISPR/Cas9., Results: Our results suggest that 1,25[OH]2D3/VDR stimulation supports differentiation of the colonic epithelium and that impaired 1,25[OH]2D3/VDR signalling thereby may compromise the structure of the intestinal epithelial barrier, leading to flares of UC. Furthermore, a transcriptional response to VDR activity was observed primarily in fully differentiated cells at the top of the colonic crypt, and this response was reduced during flares of UC., Conclusions: We identified an important role of vitamin D signalling in supporting differentiated cell states in the human colonic epithelium, and thereby maintenance of the intestinal barrier integrity. This makes the vitamin D-VDR signalling axis an interesting target for therapeutic efforts to achieve and maintain remission in patients with UC., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

The intestinal epithelium ensures uptake of vital nutrients and acts as a barrier between luminal contents and the underlying immune system. In inflammatory bowel diseases, such as ulcerative colitis (UC), this barrier is compromised, and patients experience debilitating symptoms. Here, we perform single-cell RNA profiling of epithelial cells and outline patterns of cell fate decisions in healthy individuals and UC patients. We demonstrate that patterns of hierarchical behavior are altered in UC patients and identify unique cellular states associated with Janus kinase/signal transducer and activator of transcription (JAK/STAT) activation in ulcerated and non-ulcerated areas of the colonic epithelium. These transcriptional changes could be recapitulated in human colonic organoids, wherein cytokine-mediated activation of JAK/STAT led to the emergence of cell populations with augmented regenerative properties. Altogether, our findings indicate that intricate relationships between epithelial and cytokine signaling regulate cell fate during epithelial tissue regeneration in humans and have important implications for the understanding of UC biology., Competing Interests: Declaration of interests T.S. is an inventor on several patents related to organoids., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mindful eating (ME) has been linked to improvement in binge eating disorder, but this approach in obesity management has shown conflicting results. Our aim was to assess the effect of ME associated with moderate energy restriction (MER) on weight loss in women with obesity. Metabolic parameters, dietary assessment, eating behaviour, depression, anxiety and stress were also evaluated. A total of 138 women with obesity were randomly assigned to three intervention groups: ME associated with MER (ME + MER), MER and ME, and they were followed up monthly for 6 months. ME + MER joined seven monthly mindfulness-based intervention group sessions each lasting 90 min and received an individualised food plan with MER (deficit of 2092 kJ/d - 500 kcal/d). MER received an individualised food plan with MER (deficit of 2092 kJ/d - 500 kcal/d), and ME joined seven monthly mindfulness-based intervention group sessions each lasting 90 min. Seventy patients completed the intervention. Weight loss was significant, but no statistically significant difference was found between the groups. There was a greater reduction in uncontrolled eating in the ME group than in the MER group and a greater reduction in emotional eating in the ME group than in both the MER and the ME + MER groups. No statistically significant differences were found in the other variables evaluated between groups. The association between ME with energy restriction did not promote greater weight loss than ME or MER.

During intestinal organogenesis, equipotent epithelial progenitors mature into phenotypically distinct stem cells that are responsible for lifelong maintenance of the tissue. While the morphological changes associated with the transition are well characterized, the molecular mechanisms underpinning the maturation process are not fully understood. Here, we leverage intestinal organoid cultures to profile transcriptional, chromatin accessibility, DNA methylation, and three-dimensional (3D) chromatin conformation landscapes in fetal and adult epithelial cells. We observed prominent differences in gene expression and enhancer activity, which are accompanied by local changes in 3D organization, DNA accessibility, and methylation between the two cellular states. Using integrative analyses, we identified sustained Yes-Associated Protein (YAP) transcriptional activity as a major gatekeeper of the immature fetal state. We found the YAP-associated transcriptional network to be regulated at various levels of chromatin organization and likely to be coordinated by changes in extracellular matrix composition. Together, our work highlights the value of unbiased profiling of regulatory landscapes for the identification of key mechanisms underlying tissue maturation.

Glioblastoma (GBM) stem cells (GSCs) display phenotypic and molecular features reminiscent of normal neural stem cells and exhibit a spectrum of cell cycle states (dormant, quiescent, proliferative). However, mechanisms controlling the transition from quiescence to proliferation in both neural stem cells (NSCs) and GSCs are poorly understood. Elevated expression of the forebrain transcription factor FOXG1 is often observed in GBMs. Here, using small-molecule modulators and genetic perturbations, we identify a synergistic interaction between FOXG1 and Wnt/β-catenin signaling. Increased FOXG1 enhances Wnt-driven transcriptional targets, enabling highly efficient cell cycle re-entry from quiescence; however, neither FOXG1 nor Wnt is essential in rapidly proliferating cells. We demonstrate that FOXG1 overexpression supports gliomagenesis in vivo and that additional β-catenin induction drives accelerated tumor growth. These data indicate that elevated FOXG1 cooperates with Wnt signaling to support the transition from quiescence to proliferation in GSCs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Obesity is a chronic disease resulting from multifactorial causes mainly related to lifestyle (sedentary lifestyle, inadequate eating habits) and to other conditions such as genetic, hereditary, psychological, cultural, and ethnic factors. The weight loss process is slow and complex, and involves lifestyle changes with an emphasis on nutritional therapy, physical activity practice, psychological interventions, and pharmacological or surgical treatment. Because the management of obesity is a long-term process, it is essential that the nutritional treatment contributes to the maintenance of the individual's global health. The main diet-related causes associated with excess weight are the high consumption of ultraprocessed foods, which are high in fats, sugars, and have high energy density; increased portion sizes; and low intake of fruits, vegetables, and grains. In addition, some situations negatively interfere with the weight loss process, such as fad diets that involve the belief in superfoods, the use of teas and phytotherapics, or even the avoidance of certain food groups, as has currently been the case for foods that are sources of carbohydrates. Individuals with obesity are often exposed to fad diets and, on a recurring basis, adhere to proposals with promises of quick solutions, which are not supported by the scientific literature. The adoption of a dietary pattern combining foods such as grains, lean meats, low-fat dairy, fruits, and vegetables, associated with an energy deficit, is the nutritional treatment recommended by the main international guidelines. Moreover, an emphasis on behavioral aspects including motivational interviewing and the encouragement for the individual to develop skills will contribute to achieve and maintain a healthy weight. Therefore, this Position Statement was prepared based on the analysis of the main randomized controlled studies and meta-analyses that tested different nutrition interventions for weight loss. Topics in the frontier of knowledge such as gut microbiota, inflammation, and nutritional genomics, as well as the processes involved in weight regain, were included in this document. This Position Statement was prepared by the Nutrition Department of the Brazilian Association for the Study of Obesity and Metabolic Syndrome (ABESO), with the collaboration of dietitians from research and clinical fields with an emphasis on strategies for weight loss., (© 2023. The Author(s).)

Brain organoids are becoming increasingly relevant to dissect the molecular mechanisms underlying psychiatric and neurological conditions. The in vitro recapitulation of key features of human brain development affords the unique opportunity of investigating the developmental antecedents of neuropsychiatric conditions in the context of the actual patients' genetic backgrounds. Specifically, multiple strategies of brain organoid (BO) differentiation have enabled the investigation of human cerebral corticogenesis in vitro with increasing accuracy. However, the field lacks a systematic investigation of how closely the gene co-expression patterns seen in cultured BO from different protocols match those observed in fetal cortex, a paramount information for ensuring the sensitivity and accuracy of modeling disease trajectories. Here we benchmark BO against fetal corticogenesis by integrating transcriptomes from in-house differentiated cortical BO (CBO), other BO systems, human fetal brain samples processed in-house, and prenatal cortices from the BrainSpan Atlas. We identified co-expression patterns and prioritized hubs of human corticogenesis and CBO differentiation, highlighting both well-preserved and discordant trends across BO protocols. We evaluated the relevance of identified gene modules for neurodevelopmental disorders and psychiatric conditions finding significant enrichment of disease risk genes especially in modules related to neuronal maturation and synapsis development. The longitudinal transcriptomic analysis of CBO revealed a two-step differentiation composed of a fast-evolving phase, corresponding to the appearance of the main cell populations of the cortex, followed by a slow-evolving one characterized by milder transcriptional changes. Finally, we observed heterochronicity of differentiation across BO models compared to fetal cortex. Our approach provides a framework to directly compare the extent of in vivo/in vitro alignment of neurodevelopmentally relevant processes and their attending temporalities, structured as a resource to query for modeling human corticogenesis and the neuropsychiatric outcomes of its alterations., (© 2022. The Author(s).)

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio , as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.

Organs are anatomically compartmentalised to cater for specialised functions. In the small intestine (SI), regionalisation enables sequential processing of food and nutrient absorption. While several studies indicate the critical importance of non-epithelial cells during development and homeostasis, the extent to which these cells contribute to regionalisation during morphogenesis remains unexplored. Here, we identify a mesenchymal-epithelial crosstalk that shapes the developing SI during late morphogenesis. We find that subepithelial mesenchymal cells are characterised by gradients of factors supporting Wnt signalling and stimulate epithelial growth in vitro. Such a gradient impacts epithelial gene expression and regional villus formation along the anterior-posterior axis of the SI. Notably, we further provide evidence that Wnt signalling directly regulates epithelial expression of Sonic Hedgehog (SHH), which, in turn, acts on mesenchymal cells to drive villi formation. Taken together our results uncover a mechanistic link between Wnt and Hedgehog signalling across different cellular compartments that is central for anterior-posterior regionalisation and correct formation of the SI., (© 2022. The Author(s).)

Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

During mitosis, chromatin condensation is accompanied by a global arrest of transcription. Recent studies suggest transcriptional reactivation upon mitotic exit occurs in temporally coordinated waves, but the underlying regulatory principles have yet to be elucidated. In particular, the contribution of sequence-specific transcription factors (TFs) remains poorly understood. Here we report that Brn2, an important regulator of neural stem cell identity, associates with condensed chromatin throughout cell division, as assessed by live-cell imaging of proliferating neural stem cells. In contrast, the neuronal fate determinant Ascl1 dissociates from mitotic chromosomes. ChIP-seq analysis reveals that Brn2 mitotic chromosome binding does not result in sequence-specific interactions prior to mitotic exit, relying mostly on electrostatic forces. Nevertheless, surveying active transcription using single-molecule RNA-FISH against immature transcripts reveals differential reactivation kinetics for key targets of Brn2 and Ascl1, with transcription onset detected in early (anaphase) versus late (early G1) phases, respectively. Moreover, by using a mitotic-specific dominant-negative approach, we show that competing with Brn2 binding during mitotic exit reduces the transcription of its target gene Nestin Our study shows an important role for differential binding of TFs to mitotic chromosomes, governed by their electrostatic properties, in defining the temporal order of transcriptional reactivation during mitosis-to-G1 transition., (© 2021 Soares et al.; Published by Cold Spring Harbor Laboratory Press.)

Point mutations within the histone H3.3 are frequent in aggressive childhood brain tumors known as pediatric high-grade gliomas (pHGGs). Intriguingly, distinct mutations arise in discrete anatomical regions: H3.3-G34R within the forebrain and H3.3-K27M preferentially within the hindbrain. The reasons for this contrasting etiology are unknown. By engineering human fetal neural stem cell cultures from distinct brain regions, we demonstrate here that cell-intrinsic regional identity provides differential responsiveness to each mutant that mirrors the origins of pHGGs. Focusing on H3.3-G34R, we find that the oncohistone supports proliferation of forebrain cells while inducing a cytostatic response in the hindbrain. Mechanistically, H3.3-G34R does not impose widespread transcriptional or epigenetic changes but instead impairs recruitment of ZMYND11, a transcriptional repressor of highly expressed genes. We therefore propose that H3.3-G34R promotes tumorigenesis by focally stabilizing the expression of key progenitor genes, thereby locking initiating forebrain cells into their pre-existing immature state., Competing Interests: Declaration of interests S.M.P. is a founder and shareholder of Cellinta Ltd., a biotechnology startup that is developing cancer therapeutics, including glioblastoma. S.M.P. is also an inventor on a University of Edinburgh patent related to NSC culture methods (WO2005121318A3). The other authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Testosterone (T) and its 17-α epimer, epitestosterone (EpiT), are described as having non-classical effects in addition to their classical androgen actions via the intracellular androgen receptor (iAR). The actions of these androgens play an essential role in triggering factors that shift Sertoli cells from the proliferation phase to the maturation phase. This process is essential for successful spermatogenesis and normal fertility. The aim of this work was to investigate the difference between T and EpiT effects in normal and in chemically castrated Wistar rats. We also tested the effects of these hormones when the iAR-dependent pathways were inhibited by the antiandrogen flutamide. Rats were chemically castrated on postnatal day (pnd) 5 using EDS, a cytotoxic agent that promotes apoptosis of Leydig cells, reducing androgen levels. Then, animals received replacement with T or EpiT and were treated or not with flutamide from pnd 6 to pnd 13 or 20 and were euthanized on pnd 14 and 21. Animals treated with EpiT and flutamide had lower body weight overall. Epididymis weight was also reduced in animals treated with EpiT and flutamide. Flutamide per se reduced epididymis weight at both ages (pnd 14 and 21). Testicular weight and the testicular/body weight ratio were reduced in EDS animals, and flutamide further reduced this weight in animals which received T replacement. EDS administration reduced mRNA levels of both AMH (anti-Müllerian hormone) and its receptor, AMHR2, at pnd 14. In the testes of flutamide-treated animals, EpiT reduced AMH, and both T and EpiT replacement diminished AMHR2 mRNA expression also on pnd 14. EDS decreased iAR expression, and androgen replacement did not change this effect on pnd 21. In rats receiving flutamide, only those also receiving T and EpiT replacement exhibited decreased iAR expression. An increase in connexin 43 expression was observed in animals treated with EpiT without flutamide, whereas in rats treated with flutamide, both hormones were ineffective to increase connexin 43 expression reduced by EDS. Our results suggest that EpiT has an antiandrogen effect on androgen-sensitive tissues such as the epididymis. Nonetheless, the effects of T and EpiT on testicular development parameters are similar. Both hormones may act through their iAR-independent non-classical pathway, regulating AMH and AMHR2, as well as iAR expression., (Copyright © 2020 Elsevier B.V. All rights reserved.)

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells., Competing Interests: PD, BS, KM, GM, EO, NQ, AT, ML, CP, MK, CB, RF, RB, GM, Av, ST, JK, SP No competing interests declared, AJ employed by Integrated DNA Technologies (IDT), who sells reagents similar to some described herein. IDT is, however, not a publicly traded company and the authors do not own any shares or equity in IDT. No other authors have any financial interests or relationships with IDT; nor do they own any shares or equity, MB Mark A Behlke: employed by Integrated DNA Technologies (IDT), who sells reagents similar to some described herein. IDT is, however, not a publicly traded company and the authors do not own any shares or equity in IDT. No other authors have any financial interests or relationships with IDT; nor do they own any shares or equity, (© 2018, Dewari et al.)

Experimental tools for precise manipulation of mammalian genomes enable reverse genetic approaches to explore biology and disease. Powerful genome editing technologies built upon designer nucleases, such as CRISPR/Cas9, have recently emerged. Parallel progress has been made in methodologies for the expansion and differentiation of human pluripotent and tissue stem cells. Together these innovations provide a remarkable new toolbox for human cellular genetics and are opening up vast opportunities for discoveries and applications across the breadth of life sciences research. In this chapter, we review the emergence of genome editing technologies and how these are being deployed in studies of human neurobiology, neurological disease, and neuro-oncology. We focus our discussion on CRISPR/Cas9 and its application in studies of human neural stem and progenitor cells.

Spinal cord injury (SCI) is a devastating neurologic disorder with significant impacts on quality of life, life expectancy, and economic burden. Although there are no fully restorative treatments yet available, several animal and small-scale clinical studies have highlighted the therapeutic potential of cellular interventions for SCI. Mesenchymal stem cells (MSCs)-which are conventionally isolated from the bone marrow-recently emerged as promising candidates for treating SCI and have been shown to provide trophic support, ameliorate inflammatory responses, and reduce cell death following the mechanical trauma. Here we evaluated the human skin as an alternative source of adult MSCs suitable for autologous cell transplantation strategies for SCI. We showed that human skin-derived MSCs (hSD-MSCs) express a range of neural markers under standard culture conditions and are able to survive and respond to neurogenic stimulation in vitro. In addition, using histological analysis and behavioral assessment, we demonstrated as a proof-of-principle that hSD-MSC transplantation reduces the severity of tissue loss and facilitates locomotor recovery in a rat model of SCI. Altogether, the study provides further characterization of skin-derived MSC cultures and indicates that the human skin may represent an attractive source for cell-based therapies for SCI and other neurological disorders. Further investigation is needed to elucidate the mechanisms by which hSD-MSCs elicit tissue repair and/or locomotor recovery.

Glioblastoma multiforme (GBM) is an aggressive brain tumor driven by cells with hallmarks of neural stem (NS) cells. GBM stem cells frequently express high levels of the transcription factors FOXG1 and SOX2. Here we show that increased expression of these factors restricts astrocyte differentiation and can trigger dedifferentiation to a proliferative NS cell state. Transcriptional targets include cell cycle and epigenetic regulators (e.g., Foxo3 , Plk1 , Mycn , Dnmt1 , Dnmt3b , and Tet3 ). Foxo3 is a critical repressed downstream effector that is controlled via a conserved FOXG1/SOX2-bound cis -regulatory element. Foxo3 loss, combined with exposure to the DNA methylation inhibitor 5-azacytidine, enforces astrocyte dedifferentiation. DNA methylation profiling in differentiating astrocytes identifies changes at multiple polycomb targets, including the promoter of Foxo3 In patient-derived GBM stem cells, CRISPR/Cas9 deletion of FOXG1 does not impact proliferation in vitro; however, upon transplantation in vivo, FOXG1 -null cells display increased astrocyte differentiation and up-regulate FOXO3. In contrast, SOX2 ablation attenuates proliferation, and mutant cells cannot be expanded in vitro. Thus, FOXG1 and SOX2 operate in complementary but distinct roles to fuel unconstrained self-renewal in GBM stem cells via transcriptional control of core cell cycle and epigenetic regulators., (© 2017 Bulstrode et al.; Published by Cold Spring Harbor Laboratory Press.)

Folate deficiency and hyperhomocysteinemia have long been associated with developmental anomalies, particularly neural tube defects and neurocristopathies-a group of diverse disorders that result from defective growth, differentiation, and migration of neural crest (NC) cells. However, the exact mechanisms by which homocysteine (Hcys) and/or folate deficiencies disrupt NC development are still poorly understood in mammals. In this work, we employed a well-defined culture system to investigate the effects of Hcys and folic acid (FA) supplementation on the morphogenetic processes of murine NC cells in vitro. We demonstrated that Hcys increases outgrowth and proliferation of cephalic NC cells and impairs their differentiation into smooth muscle cells. In addition, we showed that FA alone does not directly affect the developmental dynamics of the cephalic NC cells but is able to prevent the Hcys-induced effects. Our results, therefore, suggest that elevated Hcys levels per se cause dysmorphogenesis of the cephalic NC and might contribute to neurocristopathies in mammalian embryos.

Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagated in vitro as adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci ( Rosa26 and AAVS1 ); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry ); and (4) engineering of glioma mutations ( TP53 deletion; H3F3A point mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis., (© 2017. Published by The Company of Biologists Ltd.)

Even though it is a rare event, most associations of thyroid carcinoma with subacute thyroiditis described in the literature are related to its granulomatous form (Quervain's thyroiditis). We present a patient with subacute lymphocytic thyroiditis (painless thyroiditis) and papillary thyroid cancer that was first suspected in an initial ultrasound evaluation. A 30-year old female patient who was referred to the emergency room due to hyperthyroidism symptoms was diagnosed with painless thyroiditis established by physical examination and laboratory findings. With the presence of a palpable painless thyroid nodule an ultrasound was prescribed and the images revealed a suspicious thyroid nodule, microcalcification focus in the heterogeneous thyroid parenquima and cervical lymphadenopathy. Fine needle aspiration biopsy was taken from this nodule; cytology was assessed for compatibility with papillary thyroid carcinoma. Postsurgical pathology evaluation showed a multicentric papillary carcinoma and lymphocytic infiltration. Subacute thyroiditis, regardless of type, may produce transitory ultrasound changes that obscure the coexistence of papillary carcinoma. Due to this, initial thyroid ultrasound evaluation should be delayed until clinical recovery. We recommended a thyroid ultrasound exam for initial evaluation of painless thyroiditis, particularly in patients with palpable thyroid nodule. Further cytological examination is recommended in cases presenting with suspect thyroid nodule and/or non-nodular hypoechoic (> 1 cm) or heterogeneous areas with microcalcification focus.

Epidermal neural crest stem cells (EPI-NCSCs), which reside in the bulge of hair follicles, are attractive candidates for several applications in cell therapy, drug screening and tissue engineering. As suggested remnants of the embryonic neural crest (NC) in an adult location, EPI-NCSCs are able to generate a wide variety of cell types and are readily accessible by a minimally invasive procedure. Since the combination of epidermal growth factor (EGF) and fibroblast growth factor type 2 (FGF2) is mitogenic and promotes the neuronal commitment of various stem cell populations, we examined its effects in the proliferation and neuronal potential of mouse EPI-NCSCs. By using a recognized culture protocol of bulge whiskers follicles, we were able to isolate a population of EPI-NCSCs, characterized by the migratory potential, cell morphology and expression of phenotypic markers of NC cells. EPI-NCSCs expressed neuronal, glial and smooth muscle markers and exhibited the NC-like fibroblastic morphology. The treatment with the combination EGF and FGF2, however, increased their proliferation rate and promoted the acquisition of a neuronal-like morphology accompanied by reorganization of neural cytoskeletal proteins βIII-tubulin and nestin, as well as upregulation of the pan neuronal marker βIII-tubulin and down regulation of the undifferentiated NC, glial and smooth muscle cell markers. Moreover, the treatment enhanced the response of EPI-NCSCs to neurogenic stimulation, as evidenced by induction of GAP43, and increased expression of Mash-1 in neuron-like cell, both neuronal-specific proteins. Together, the results suggest that the combination of EGF-FGF2 stimulates the proliferation and improves the neuronal potential of EPI-NCSCs similarly to embryonic NC cells, ES cells and neural progenitor/stem cells of the central nervous system and highlights the advantage of using EGF-FGF2 in neuronal differentiation protocols., (Copyright © 2014 Elsevier Inc. All rights reserved.)